Generation, characterization, and isolation of neuroepithelial stem cells and lineage restricted intermediate precursor

ABSTRACT

Multipotent neuroepithelial stem cells and lineage-restricted oligodendrocyte-astrocyte precursor cells are described. The neuroepithelial stem cells are capable of self-renewal and of differentiation into neurons, astrocytes, and oligodendrocytes. The oligodendrocyte-astrocyte precursor cells are derived from neuroepithelial stem cells, are capable of self-renewal, and can differentiate into oligodendrocytes and astrocytes, but not neurons. Methods of generating, isolating, and culturing such neuroepithelial stem cells and oligodendrocyte-astrocyte precursor cells are also disclosed.

BACKGROUND OF THE INVENTION

[0001] This invention relates to multipotent neuroepithelial stem cells,lineage-restricted intermediate precursor cells, and methods of makingthereof. More particularly, the invention relates to neuroepithelialstem cells that retain the capabilities of self-renewal anddifferentiation into neurons, astrocytes, and oligodendrocytes. Further,the invention relates to oligodendrocyte-astrocyte-restricted precursorcells that are capable of self-renewal and differentiation intoastrocytes and oligodendrocytes, but not neurons. Methods of generating,isolating, and culturing such neuroepithelial stem cells andoligodendrocyte-astrocyte precursor cells are also described.

[0002] Multipotent cells with the characteristics of stem cells havebeen identified in several regions of the central nervous system and atseveral developmental stages. F. H. Gage et al., Isolation,Characterization and Use of Stem Cells from the CNS, 18 Ann. Rev.Neurosci. 159-92 (1995); M. Marvin & R. McKay, Multipotential Stem Cellsin (1995); M. Marvin & R. McKay, Multipotential Stem Cells in theVertebrate CNS, 3 Semin. Cell. Biol. 401-11 (1992); R. P. Skoff, TheLineages of Neuroglial Cells, 2 The Neuroscientist 335-44 (1996). Thesecells, often referred to as neuroepithelial stem cells (NEP cells), havethe capacity to undergo self renewal and to differentiate into neurons,oligodendrocytes, and astrocytes, thus representing multipotent stemcells. A. A. Davis & S. Temple, A Self-Renewing Multipotential Stem Cellin Embryonic Rat Cerebral Cortex, 362 Nature 363-72 (1994); A. G. Grittiet al., Multipotential Stem Cells from the Adult Mouse Brain Proliferateand Self-Renew in Response to Basic Fibroblast Growth Factor, 16 J.Neurosci. 1091-1100 (1996); B. A. Reynolds et al., A MultipotentEGF-Responsive Striatal Embryonic Progenitor Cell Produces Neurons andAstrocytes, 12 J. Neurosci. 4565-74 (1992); B. A. Reynolds & S. Weiss,Clonal and Population Analyses Demonstrate that an EGF-ResponsiveMammalian Embryonic CNS Precursor is a Stem Cell, 175 DevelopmentalBiol. 1-13 (1996); B. P. Williams et al., The Generation of Neurons andOligodendrocytes from a Common Precursor Cell, 7 Neuron 685-93 (1991).

[0003] The nervous system also contains precursor cells with restricteddifferentiation potentials. T. J. Kilpatrick & P. F. Bartlett, ClonedMultipotential Precursors from the Mouse Cerebrum Require FGF-2, WhereasGlial Restricted Precursors are Stimulated with Either FGF-2 or EGF, 15J. Neurosci. 3653-61 (1995); J. Price et al., Lineage Analysis in theVertebrate Nervous System by Retrovirus-Mediated Gene Transfer, 84Developmental Biol. 156-60 (1987); B. A. Reynolds et al., supra; B. A.Reynolds & S. Weiss, supra; B. Williams, Precursor Cell Types in theGerminal Zone of the Cerebral Cortex, 17 BioEssays 391-93 (1995); B. P.Williams et al., supra. The relationship between multipotent stem cellsand lineage restricted precursor cells is still unclear. In principal,lineage restricted cells could be derived from multipotent cells, butthis is still a hypothetical possibility in the nervous system with nodirect experimental evidence.

[0004] During development, the neuroepithelial cells that comprise thecaudal neural tube differentiate into neurons and glia. Neurons arisefrom neuroepithelial precursors first and eventually develop uniquephenotypes defined by their trophic requirements, morphology, andfunction. Motoneurons are among the first neurons to develop. V.Hamburger, The Mitotic Patterns in the Spinal Cord of the Chick Embryoand Their Relationship to the Histogenic Process, 88 J. Comp. Neurol.221-84 (1948); H. O. Nornes & G. D. Das, Temporal Pattern ofNeurogenesis in the Spinal Cord of Rat. 1. Time and Sites of Origin andMigration and Settling Patterns of Neuroblasts, 73 Brain Res. 121-38(1974); J. Altman & S. Bayer, The Development of the Rat Spinal Cord, 85Adv. Anat. Embryol. Cell Biol. 32-46 (1984); P. E. Phelps et al.,Generation Patterns of Four Groups of Cholinergic Neurons in RatCervical Spinal Cord: A Combined Tritiated Thymidine Autoradiographicand Choline Acetyltransferase Immunocytochemical Study, 273 J. Comp.Neurol. 459-72 (1988); P. E. Phelps et al., Embryonic Development ofFour Subsets of Cholinergic Neurons in Rat Cervical Spinal Cord, 291 J.Comp. Neurol. 9-26 (1990). Motoneurons can be distinguished from otherneurons present in the spinal cord by their position and the expressionof a number of specific antigens. E. W. Chen & A. Y. Chiu, Early Stagesin the Development of Spinal Motor Neurons, 320 J. Comp. Neurol. 291-303(1992). Tag-1, J. Dodd et al., Spatial Regulation of Axonal GlycoproteinExpression on Subsets of Embryonic Spinal Neurons, 1 Neuron 105-16(1988), islet-1, J. Erickson et al., Early Stages of Motor NeuronDifferentiation Revealed by Expression of Homeobox Gene Islet-1, 256Science 1555-59 (1992), and p75, W. Camu & C. E. Henderson, Purificationof Embryonic Rat Motorneurons by Panning on a Monoclonal Antibody to theLow-Affinity NGF Receptor, 44 J. Neurosci. 59-70 (1992), are expresseduniquely on rat and chick motoneurons early in their development, butare not detectable on other spinal cord cells and, therefore, may serveto distinguish motoneurons from other neural tube cells. Astrocytes,characterized by glial fibrillary acidic protein (GFAP)immunoreactivity, appear soon after; GFAP staining is seen at embryonicday 16 (E16). M. Hirano & J. E. Goldman, Gliogenesis in the Rat SpinalCord: Evidence for the Origin of Astrocytes and Oligodendrocytes fromRadial Precursors, 21 J. Neurosci. Res. 155-67 (1988). Astrocytic cellsproliferate and populate the gray and white matter of the spinal cord,and both type 1 and type 2 astrocytes have been identified in the spinalcord. B. C. Warf et al., Evidence for the Ventral Origin ofOligodendrocytic Precursors in the Rat Spinal Cord, 11 J. Neurosci.2477-88 (1991). Oligodendrocytes appear later and are first detectedaround birth, though oligodendrocyte precursors may be present as earlyas E14 based on platelet derived growth factor alpha-receptor (PDGFRA)expression and culture assays. N. P. Pringle & W. D. Richardson, ASingularity of PDGF Alpha-Receptor Expression in the Dorsoventral Axisof the Neural Tube May Define the Origin of the Oligodendrocyte Lineage,117 Development 525-33 (1993); B. C. Warf et al., supra.

[0005] As will be shown herein, NEP cells grow on fibronectin andrequire fibroblast growth factor (FGF) and an as yet uncharacterizedcomponent present in chick embryo extract (CEE) to proliferate andmaintain an undifferentiated phenotype in culture. The growthrequirements of NEP cells are different from neurospheres isolated fromE14.5 cortical ventricular zone cells. B. A. Reynolds et al., supra; B.A. Reynolds & S. Weiss, supra; WO 9615226; WO 9615224; WO 9609543; WO9513364; WO 9416718; WO 9410292; WO 9409119. Neurospheres grow insuspension culture and do not require CEE or FGF, but are dependent onepidermal growth factor (EGF) for survival. FGF itself is not sufficientfor long term growth of neurospheres, though FGF may support theirgrowth transiently. The presently described NEP cells grow in adherentculture, are FGF dependent, do not express detectable levels of EGFreceptors, and are isolated at a stage of embryonic development prior towhich it has been possible to isolate neurospheres. Thus, NEP cells mayrepresent a multipotent precursor characteristic of the brain stem andspinal cord, while neurospheres may represent a stem cell morecharacteristic of the cortex.

[0006] U.S. Pat. No. 5,589,376, to D. J. Anderson and D. L. Stemple,discloses mammalian neural crest stem cells and methods of isolation andclonal propagation thereof, but fails to disclose cultured NEP cells,cultured lineage restricted precursor cells, and methods of generating,isolating, and culturing thereof. Neural crest cells differentiate intoneurons and glia of the peripheral nervous system (PNS), whereas thepresent neuroepithelial stem cells differentiate into neurons and gliaof the central nervous system (CNS).

[0007] The present invention is necessary to understand how multipotentneuroepithelial stem cells become restricted to the variousneuroepithelial derivatives. In particular, culture conditions thatallow the growth and self-renewal of mammalian neuroepithelial stemcells are desirable so that the particulars of the development of thesemammalian stem cells can be ascertained. This is desirable because anumber of tumors of neuroepithelial derivatives exist in mammals,particularly humans. Knowledge of mammalian neuroepithelial stem celldevelopment is therefore needed to understand these disorders in humans.Additionally, the ability to isolate and grow mammalian neuroepithelialstem cells in vitro allows for the possibility of using such stem cellsto treat neurological disorders in mammals, particularly humans.Further, such mammalian neuroepithelial stem cells can be usedtherapeutically for treatment of certain diseases, e.g. Parkinson'sDisease, such as by transplantation of such cells into an afflictedindividual. Moreover, such cells can still further be used for thediscovery of genes and drugs that are useful for treating certaindiseases. For example, novel genes can be identified by differentialdisplay or subtractive hybridization or other screening strategies.Still further, pure NEP stem cell populations according to the presentinvention can be used to generate and screen antibodies that arespecific for these specific cells.

[0008] In view of the foregoing, it will be appreciated that isolatedpopulations of mammalian neuroepithelial stem cells and lineagerestricted glial precursor cells and methods of generating, isolating,and culturing such cells would be a significant advancement in the art.

BRIEF SUMMARY OF THE INVENTION

[0009] It is an object of the present invention to provide isolated(pure) populations of mammalian neuroepithelial stem cells and theirprogeny in feeder-cell-independent adherent cultures.

[0010] It is also an object of the invention to provide populations ofmammalian lineage-restricted glial precursor cells and their progeny infeeder-cell-independent adherent cultures.

[0011] It is another object of the invention to provide methods ofgenerating, isolating, and culturing mammalian neuroepithelial stemcells and lineage restricted glial precursor cells and their progeny.

[0012] It is still another object of the invention to provide methodsfor the growth and regeneration of neuroepithelial stem cells andlineage restricted glial precursor cells in feeder-cell-independentadherent cultures.

[0013] It is yet another object of the invention to provide a method forthe generation of lineage restricted glial precursor cells fromneuroepithelial stem cells.

[0014] It is a still further object of the invention to provide puredifferentiated populations of cells derived from neuroepithelial stemcells and lineage restricted glial precursor cells.

[0015] It is yet another object of the invention to provide cDNA andcDNA libraries from pure differentiated populations derived fromneuroepithelial stem cells and lineage restricted glial precursor cells.

[0016] It is still another object of the invention to provide antibodiesspecific to NEP cells and lineage restricted glial precursor cells.

[0017] These and other objects can be achieved by providing an isolated,pure population of mammalian CNS neuroepithelial stem cells wherein thecells are capable of self-renewal and proliferation in afeeder-cell-independent adherent culture medium and of differentiationto CNS neuronal or glial cells. Preferably, such neuroepithelial stemcells express nestin, but do not express polysialated neural celladhesion molecule, glial fibrillary acidic protein, sulfatide,neurofilament, choline acetyl transferase, intermediate filament,ganglioside, or galactocerebroside. It is also preferred that such CNSneuronal cells do not express intermediate filament and neurofilament68, choline acetyl transferase, glial fibrillary acidic protein,ganglioside, sulfatide, or galactocerebroside. The neuroepithelial stemcells preferably are further capable of differentiation toglial-restricted precursor cells. Such glial-restricted precursor cellsare preferably capable of self-renewal and proliferation in afeeder-cell-independent adherent culture medium and of differentiationto CNS glial cells but not to CNS neuronal cells. These glial-restrictedprecursor cells preferably express nestin and ganglioside, but do notexpress glial fibrillary acidic protein, sulfatide, orgalactocerebroside.

[0018] Another illustrative embodiment of the invention comprises anisolated, pure population of mammalian CNS glial-restricted precursorcells, wherein the glial-restricted precursor cells are capable ofself-renewal and proliferation in a feeder-cell-independent adherentculture medium and of differentiation to CNS glial cells but not to CNSneuronal cells.

[0019] Still another illustrative embodiment of the invention comprisesa method of isolating a pure population of mammalian CNS neuroepithelialstem cells wherein the cells are capable of self-renewal infeeder-cell-independent adherent culture medium and of differentiationto CNS neuronal or glial cells, comprising the steps of:

[0020] (a) removing a neural tube from a mammalian embryo at a stage ofembryonic development after closure of the neural tube but prior todifferentiation of cells in the neural tube;

[0021] (b) dissociating cells comprising the neural tube removed fromthe mammalian embryo;

[0022] (c) plating the dissociated cells in feeder-cell-independentculture on a substratum and in a medium configured for supportingadherent growth of the neuroepithelial stem cells, wherein the mediumcomprises effective amounts of fibroblast growth factor and chick embryoextract; and

[0023] (d) incubating the plated cells at a temperature and in anatmosphere conducive to growth of the neuroepithelial stem cells.

[0024] Yet another illustrative embodiment of the invention comprises amethod of isolating a pure population of mammalian CNS glial-restrictedprecursor cells wherein the cells are capable of self-renewal infeeder-cell-independent adherent culture medium and of differentiationto CNS glial cells but not CNS neuronal cells, comprising the steps of:

[0025] (a) isolating a population of mammalian CNS neuroepithelial stemscells;

[0026] (b) incubating the neuroepithelial stem cells in a mediumconfigured for supporting growth of the neuroepithelial stem cellsexcept for lacking an effective amount of chick embryo extract for aperiod of time sufficient for the cells to begin differentiating;

[0027] (c) subjecting the incubated cells to specific antibody captureusing an antibody characteristic of glial-restricted precursor cells toresult in a captured subpopulation of cells; and

[0028] (d) incubating the captured subpopulation of cells in a mediumconfigured for supporting growth thereof comprising effective amounts offibroblast growth factor and platelet derived growth factor.

[0029] Yet another illustrative embodiment of the invention comprises amethod of generating a population of mammalian motoneurons comprisingthe steps of:

[0030] (a) isolating a population of mammalian CNS neuroepithelial stemscells;

[0031] (b) incubating the neuroepithelial stem cells in a medium thatpromotes cell proliferation and neuronal differentiation for a period oftime sufficient for the cells to begin differentiating; and

[0032] (c) isolating motoneurons from said differentiating cells. Apreferred medium comprises the use of laminin-coated plates and NEPmedium lacking an effective amount of chick embryo extract.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 shows a summary of the antigenic properties of NEP cellsand their progeny.

[0034]FIG. 2 shows a summary of the antigenic properties of multipotentNEP stem cells, glial-restricted A2B5⁺ cells (oligodendrocyte-astrocyte(O-A) progenitor) that arise from NEP cells, and oligodendrocytes andastrocytes that arise from the O-A progenitor.

DETAILED DESCRIPTION

[0035] Before the present neuroepithelial stem cells, glial-restrictedprecursor cells, and methods of making thereof are disclosed anddescribed, it is to be understood that this invention is not limited tothe particular configurations, process steps, and materials disclosedherein as such configurations, process steps, and materials may varysomewhat. It is also to be understood that the terminology employedherein is used for the purpose of describing particular embodiments onlyand is not intended to be limiting since the scope of the presentinvention will be limited only by the appended claims and equivalentsthereof.

[0036] It must be noted that, as used in this specification and theappended claims, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “an embryo” includes reference to two or moreembryos, reference to “a mitogen” includes reference to a mixture of twoor more mitogens, and reference to “a factor” includes reference to amixture of two or more factors.

[0037] In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

[0038] As used herein, “self renewal” refers to the capability of aneuroepithelial stem cell to divide to produce two daughter cells, atleast one of which is a multipotent neuroepithelial stem cell.

[0039] As used herein, “clonal density” and similar terms mean a densitysufficiently low enough to result in the isolation of single,non-impinging cells when plated in a selected culture dish. Anillustrative example of such a clonal density is about 225 cells/100 mmculture dish.

[0040] As used herein, “feeder-cell-independent adherent culture” orsimilar terms mean the growth of cells in vitro in the absence of alayer of different cells that generally are first plated on a culturedish to which the cells from the tissue of interest are then added. Infeeder cell cultures, the feeder cells provide a substratum for theattachment of cells from the tissue of interest and additionally serveas a source of mitogens and survival factors. Thefeeder-cell-independent adherent cultures herein use a chemicallydefined substratum, for example fibronectin, and mitogens or survivalfactors are provided by supplementation of the liquid culture mediumwith either purified factors or crude extracts from other cells ortissues. Therefore, in feeder-cell-independent cultures, the cells inthe culture dish are primarily cells derived from the tissue of interestand do not contain other cell types required to support the growth ofcells derived from the tissue of interest.

[0041] As used herein, “effective amount” means an amount of a growth orsurvival or other factor that is nontoxic but sufficient to provide thedesired effect and performance. For example, an effective amount of FGFas used herein means an amount selected so as to support self renewaland proliferation of NEP cells when used in combination with otheressential nutrients, factors, and the like.

[0042] The present invention is illustrated using neuroepithelial stemcells isolated from the rat. The invention encompasses all mammalianneuroepithelial stem cells and is not limited to neuroepithelial stemcells from the rat. Mammalian neuroepithelial stem cells can be isolatedfrom human and non-human primates, equines, canines, felines, bovines,porcines, ovines, lagomorphs, and the like.

[0043] The present invention concerns an embryonic spinal cord stemcell, termed NEP cell, derived from caudal neuroepithelium, thatrequires fibroblast growth factor (FGF) and chick embryo extract (CEE)to proliferate and self renew. NEP cells are characterized by: (1) theexpression of nestin, (2) the absence of lineage markers, (3) ability tobe maintained in an undifferentiated state in culture, (4) the abilityto self renew, and (5) the ability to grow in clonal culture. Underappropriate environmental conditions, NEP cells differentiate into thethree principal types of cell in the CNS, neurons, astrocytes, andoligodendrocytes. FIG. 1 presents a model for spinal corddifferentiation. This model is similar to that proposed forhematopoiesis and for differentiation of neural crest (see review by D.J. Anderson, The Neural Crest Lineage Problem: Neuropoiesis?, 3 Neuron1-12 (1989)). According to this model, NEP cells 10 represent ahomogeneous population of cells in the caudal neural tube that expressnestin (nestin⁺) but no other lineage marker (lin⁻). These cells divideand self renew in culture and generate differentiated phenotypes.Previous data have suggested intermediate dividing precursors with amore restricted potential. R. H. Miller & V. Szigeti, infra; B. C. Warfet al., supra; N. P. Pringle & W. D. Richardson, supra; J. Ray & F.Gage, Spinal Cord Neuroblasts Proliferate in Response to BasicFibroblast Growth Factor, 14 J. Neurosci. 3548-64 (1994). Suchprecursors include those precursors 14 that generate oligodendrocytes 18and type 2 astrocytes 22, bipotent astrocyte and neuronal precursors(not shown in FIG. 1), as well as neuronal progenitors that generateseveral kinds of neurons (not shown in FIG. 1). The model thereforesuggests that the multipotent precursors (NEP cells) generatedifferentiated cells (i.e., oligodendrocytes 18, type 2 astrocytes 22,type 1 astrocytes 24, neurons 26, and motoneurons 30) throughintermediate precursors. Consistent with this model are the resultspresented herein showing the existence of cells with a restrictedproliferative potential.

[0044]FIG. 1 shows that motoneurons arise from a common NEP precursor.The experiments described herein show that low affinity neurotrophinreceptor (p75) immunoreactive, choline acetyl transferase (ChAT)positive cells arise in mixed cultures along with other cells of thespinal cord. No clone consisting exclusively of p75/ChAT immunoreactivecells was identified, indicating that, at the age the clones wereanalyzed, committed motoneuron precursors were not present. Theobservation that motoneurons arise from a common NEP precursor areconsistent with results obtained in chick neural tube experiments. E.g.,M. Bronner-Fraser & S. E. Fraser, Cell Lineage Analysis SlowsMultipotentiality of Some Avian Neural Crest Cells, 355 Nature 161-64(1988). These results, together with previous observations, thereforesuggest that motoneuron differentiation involves a multipotent precursorundergoing progressive stages of commitment.

[0045] NEP cells are similar in some respects to, and yet are clearlydifferent from, neuroepithelial cultures from the myelencephalon andtelencephalon. M. Murphy et al., Fibroblast Growth Factor Stimulates theProliferation and Differentiation of Neural Precursor Cell In Vitro, 25J. Neurosci. Res. 463-75 (1990); J. Drago et al., Fibroblast GrowthFactor-Mediated Proliferation of Central Nervous System PrecursorsDepends on Endogenous Production of Insulin-like Growth Factor 1, 88Proc. Nat'l Acad. Sci. USA 2199-2203 (1991); T. J. Kilpatrick & B. F.Bartlett, Cloning and Growth of Multipotential Neural Precursors:Requirements for Proliferation and Differentiation, 10 Neuron 255-65(1993). Like those cells, NEP cells are FGF-dependent, grow as adherentcells, and require an uncharacterized component present in CEE and/orserum. The cells isolated by Murphy et al., Drago et al., and Kilpatrickand Bartlett differ from NEP cells, however, in that they do not formneurospheres. Thus, brain stem and spinal precursor cells appear to bedifferent from cortical precursors. Brain stem neuroepithelial cells arebipotent and have not been shown to differentiate into oligodendrocytes.Further, spinal cord NEP cells rapidly differentiate into astrocytes inthe presence of serum. In contrast, brain stem NEP cells remain in anundifferentiated state in the presence of serum.

[0046] NEP cells differ from neural crest stem cells in their morphologyand antigenic profile. Neural crest cells are more fibroblastic, tend tobe migratory, and avoid cell contact. S. Boisseau et al., A Mammalian InVitro Model to Study Gangliogenesis from Neural Crest Cells, 85 J.Physiol., Paris 117-22 (1991); P. G. Bannerman & D. Pleasure, ProteinGrowth Factor Requirements of Rat Neural Crest Cells, 36 J. Neurosci.Res. 46-57 (1993). NEP cells appear more flattened and epithelioid andtend to grow as tightly packed monolayers. Unlike rat neural crestcells, NEP cells do not express immunoreactivity for the low affinityneutrophin receptor (p75; Example 4). Moreover, the progeny of NEP cellsdiffer from neural crest cell derivatives. For example,GFAP-immunoreactive cells from NEP cultures do not express detectablenestin and p75 immunoreactivity (Example 6). In contrast, Schwann cells,which are glial cells of the PNS and differentiate from neural crest,express high levels of both p75 and GFAP in culture. D. L. Stemple & D.J. Anderson, Isolation of a Stem Cell for Neurons and Glia from theMammalian Neural Crest, 71 Cell 973-85 (1992). Schwann cells alsoexpress myelin markers such as O4 and P0, D. L. Stemple & D. J.Anderson, supra; M. S. Rao & D. J. Anderson, The Immortalization of aNeural Crest Stem Cell, ASCB 2098 (1994), which are not expressed byGFAP-immunoreactive cells derived from NEP cultures (e.g. Example 4).NEP cultures contain A2B5 immunoreactive cells, which subsequentlyexpress O4, GalC, and O1 immunoreactivity. Cells with this pattern ofantigen expression are not viewed as derivatives of neural crest.Further, while neural-crest-derived parasympathetic neurons express ChATimmunoreactivity in vivo, such neurons have not been described fromneural crest cultures. D. J. Anderson, supra. NEP cells, however,readily differentiate to generate large numbers of neurons co-expressingp75 and ChAT. Thus, NEP cells and neural crest stem cells aremorphologically and antigenically distinct, generate differentiatedprogeny that are phenotypically different, and therefore representdifferent stem cells.

[0047] Therefore, the NEP stem cells from the developing spinal cordcharacterized herein have some properties in common with other nervoussystem stem cells, but are clearly distinct therefrom. NEP cellsrepresent a stem cell population that undergoes self renewal anddifferentiates into all major CNS phenotypes in culture. NEP cellsdiffer from all previously identified stem cells in their cultureconditions and proliferative potential. NEP cell cultures provide alarge source of transient cells that can be sorted to obtaindifferentiated cells types.

[0048] The basal medium (NEP medium) used in the experiments describedherein comprises DMEM-F12 (GIBCO/BRL, Gaithersburg, Md.) supplementedwith 100 μg/ml transferrin (Calbiochem, San Diego, Calif.), 5 μg/mlinsulin (Sigma Chemical Co., St. Louis, Mo.), 16 μg/ml putrescine(Sigma), 20 nM progesterone (Sigma), 30 nM selenious acid (Sigma), 1mg/ml bovine serum albumin (GIBCO/BRL), plus B27 additives (GIBCO/BRL),25 ng/ml fibroblast growth factor (FGF), and 10% chick embryo extract(CEE). In general, these additives were stored as 100× concentrates at−20° C. until use. Normally, 200 ml of NEP medium was prepared with alladditives except CEE and used within two weeks of preparation. CEE wasadded to the NEP medium at the time of feeding cultured cells.

[0049] FGF and CEE were prepared as described in D. L. Stemple & D. J.Anderson, supra; M. S. Rao & D. J. Anderson, supra; L. Sommers et al.,Cellular Function of the bHLH Transcription Factor MASH1 in MammalianNeurogenesis, 15 Neuron 1245-58 (1995), hereby incorporated byreference. FGF is also available commercially (UBI).

[0050] Briefly, CEE was prepared as follows. Chick eggs were incubatedfor 11 days at 38° C. in a humidified atmosphere. Eggs were washed andthe embryos were removed and placed in a petri dish containing sterileMinimal Essential Medium (MEM with glutamine and Earle's salts)(GIBCO/BRL) at 4° C. Approximately 10 embryos each were macerated bypassage through a 30-ml syringe into a 50-ml test tube. This proceduretypically produced about 25 ml of medium. To each 25 ml was added 25 mlof MEM. The tubes were rocked at 4° C. for 1 hour. Sterile hyaluronidase(1 mg/25 g of embryo) (Sigma) was added, and the mixture was centrifugedfor 6 hours at 30,000 g. The supernate was collected, passed through a0.45 μm filter and then through a 0.22 μm filter, and stored at −80° C.until use.

[0051] Fibronectin (New York Blood Center, New York, N.Y., or Sigma) wasdiluted to a concentration of 250 μg/ml in D-PBS (GIBCO/BRL). Thefibronectin solution was applied to tissue culture dishes andimmediately withdrawn. Collagen (Biomedical Technologies, Inc.,Stoughton, Mass.) and poly-L-lysine (Sigma) were each applied to dishesat 20 μg/ml concentrations. Laminin (GIBCO/BRL or Sigma) was used at aconcentration of 50-250 μg/ml, and dishes were coated overnight. In somecases, dishes were precoated with pDL (30-70 kDa) (BiomedicalTechnologies, Inc.). The pDL was dissolved in distilled water andapplied to tissue culture plates for an hour, and then the excess pDLwas withdrawn and the plates were allowed to air dry. Plates were rinsedwith water and then allowed to dry again. The pDL-coated plates werethen coated with laminin as described above. NEP cells were dissociatedand plated on coated dishes, and their development monitored underseveral different conditions. Fibronectin was chosen as a growthsubstrate because NEP cells did not adhere to collagen or poly-L-lysineand adhered poorly to laminin. Thus, all subsequent experiments tomaintain NEP cells in culture were performed on fibronectin-coateddishes. Laminin-coated dishes were sometimes used, however, to promotedifferentiation of NEP stem cells.

EXAMPLE 1

[0052] The neural tube undergoes closure at embryonic day 10 in rats,Hamburger, supra, and earliest differentiation occurs a day later,Hamburger, supra; Nornes & Das, supra; Altman & Bayer, supra. Embryonicday 10.5 (E10.5) therefore represents the earliest time point when alarge number of undifferentiated NEP cells can be easily isolated.Sprague Dawley rat embryos were removed at E10.5 (13-22 somites) andplaced in a petri dish containing Ca/Mg-free Hanks balanced saltsolution (HBSS, GIBCO/BRL). The trunk segments of the embryos (last 10somites) were dissected using tungsten needles, rinsed, and thentransferred to fresh HBSS. Trunk segments were incubated at 4° C. in 1%trypsin solution (GIBCO/BRL) for a period of ten to twelve minutes. Thetrypsin solution was replaced with fresh HBSS containing 10% fetalbovine serum (FBS, GIBCO/BRL). The segments were gently triturated witha Pasteur pipette to release neural tubes free from surrounding somitesand connective tissue. Isolated neural tubes were transferred to a 0.05%trypsin/EDTA solution (GIBCO/BRL) for an additional period of tenminutes. Cells were dissociated by trituration and plated at highdensity in 35 mm fibronectin-coated dishes in NEP medium. Cells weremaintained at 37° C. in 5% CO₂/95% air. Cells were replated at lowdensity, i.e. ≦5000 cells per 35 mm plate, one to three days afterplating. Cells from several dishes were then harvested by trypsinization(0.05% trypsin/EDTA solution for two minutes). Cells were then pelleted,resuspended in a small volume, and counted. About 5000 cells were platedin a 35 mm dish (Corning or Nunc). For clonal analysis, cells harvestedby trypsinization were plated at a density of 50-100 cells per 35 mmdish. Individual cells were identified and located on the dish bymarking the position with a grease pencil. Cells were grown in DMEM/F12with additives, as described above, for a period ranging from 10-15days.

EXAMPLE 2

[0053] E10.5 rat neural tube cells were dissociated according to theprocedure of Example 1 except that the cells were plated at low densityand incubated in NEP medium with either acidic FGF (aFGF; 25 ng/ml),basic FGF (bFGF; 25 ng/ml), epidermal growth factor (EGF; 50 ng/ml) orno added factor for 48 hours. Cultured cells were fixed and examined byphase contrast microscopy according to methods well known in the art.Cells grown in aFGF or bFGF survived and increased in density. Incontrast, no surviving cells were seen in cultures grown without FGF orwith 50 ng/ml EGF. Thus, NEP cells require FGF for survival, and EGFdoes not support growth of NEP cells in adherent culture.

EXAMPLE 3

[0054] In this example, E10.5 rat neural tube cells were dissociatedaccording to the procedure of Example 1, and equal numbers of cells wereplated at low density in a 35 mm dish and incubated in NEP mediumcontaining bFGF (25 ng/ml) with 10% CEE or without CEE for 5 days. Thecultured cells were then fixed and examined by phase contrast microscopyaccording to methods well known in the art. In the absence of CEE, cellsgrew slowly and some cells appeared rounded and phase bright. Cellsgrown in the presence of 10% CEE appeared more homogeneous andproliferated to form a confluent monolayer. Thus, CEE was required tomaintain NEP cells in an undifferentiated state. However, CEE in itselfwas not a survival factor, and NEP cells did not survive in mediumsupplemented with CEE in the absence of exogenously added FGF. Thus, CEEcontains a component distinct from EGF that, in concert with FGF,maintains NEP cells in an undifferentiated state in culture.

EXAMPLE 4

[0055] In this example, NEP cells cultured in FGF and CEE onfibronectin-coated plates for 5 days according to the procedure ofExample 3, except for the addition of 5-bromodeoxyuridine (BrdU, 1 μMconcentration, Sigma) at day 2 to some cells, were tested byimmunocytochemistry for cell division and differentiation using avariety of antigenic markers. Nestin is a marker for undifferentiatedstem cells. U. Lendahl et al., CNS Stem Cells Express a New Class ofIntermediate Filament Protein, 60 Cell 585-95 (1990). BrdU incorporationis a marker for determining the number of dividing cells. The antiseraused and their concentrations are summarized in Table 1. All secondaryantibodies were obtained from Jackson Immunologicals (Westgrove, Pa.)and were used according to the manufacturer's instructions. Stainingprocedures were carried out as described in D. L. Stemple & D. J.Anderson, supra. Staining for cell surface antigens was carried out incultures of living cells. For neurofilament proteins, GFAP and β-IIItubulin, cells were fixed with acid-ethanol. For other intracellularantigens, cultures were fixed in 4% formaldehyde for 15 minutes. ForBrdU immunocytochemistry, cells were further permeabilized by theprocedure of S. P. Memberg & A. K. Hall, Dividing Neuron PrecursorsExpress Neuron-specific Tubulin, 27 Neurobiol. 26-43 (1995), herebyincorporated by reference. Cell cultures were incubated with theselected primary antibody in blocking buffer (PBS, 1 mg/ml bovine serumalbumin (BSA), 0.5% triton-X-100, 1% goat serum) for a period of 1 hour,rinsed with PBS, and incubated with a species-specific secondaryantibody (Jackson Immunologicals, Westgrove, Pa.) in blocking buffer foran additional hour. Cultures were rinsed with three changes of PBS.Double-labeling and triple-labeling experiments were performed bysimultaneously incubating TABLE 1 Antibody/Kind Dilution/Source AntigenRecognized Cell type Recognized Rat 401/mouse IgG 1:1/DSHB^(a)Intermediate Stem cells, oligo filament precursors anti-NCAM/mouse IgG1:3/DSHB Polysialated N-CAM Neurons anti-β-III tubulin/mouse 1:100/SigmaIntermediate Neurons IgG1 filament anti-neurofilament/mouse 1:100/SigmaNeurofilament 68 Neurons IgG2 anti-ChAT/goat IgG 1:100/Chemicon^(b)Choline acetyl Motoneurons transferase anti-glutamate/rabbit1:100/Chemicon Glutamate CSN Neurons IgG anti-GABA/rabbit IgG1:100/Chemicon Gamma amino butyric CNS Neurons acid anti-GFAP/rabbit IgG1:500/Accurate Glial fibrillary Astrocytes acid anti-A2B5/mouse IgM1:3/BMB^(c) Ganglioside oligodendrocytes and precursors anti-Gal-C/mouseIgG 1:3/BMB Galactocerebroside oligodendrocytes and precursorsanti-O4/mouse IgM 1:1/BMB Sulfatide oligodendrocytes anti-O1/mouse IgM1:3/BMB Galactocerebroside oligodendrocytes

[0056] cells in appropriate combinations of primary antibodies followedby non-cross-reactive secondary antibodies.

[0057] After 5 days in culture, all cells continued to express nestin,but did not express any other marker tested. Moreover, most of the cellshad divided and incorporated BrdU over a three-day period. These resultsindicate that the cells were dividing and were undifferentiated stemcells. NEP cells passaged at a 1:3 dilution every fifth day as adherentcultures could be maintained as nestin-immunoreactive cells that did notexpress any markers characteristic of differentiated cells over at leastthree passages. Subsequent passaging over three months maintainednestin-immunoreactive, lineage-negative cells, but in addition, a smallpercentage of GFAP-immunoreactive cells (1-5%) could be detected. Thusisolated NEP cells, which express nestin immunoreactivity and lack alllineage specific markers for neuronal and glial sublineages, could bepassaged and their numbers amplified when grown undernon-differentiation conditions.

EXAMPLE 5

[0058] The CNS consists of three major phenotypes, neurons, glia, andastrocytes, all of which express characteristic antigenic markers. Todetermine if undifferentiated, cultured NEP cells could differentiateinto CNS neurons and glia, NEP cells grown on fibronectin in NEP mediumfor 5 days according o the procedure of Example 1 were harvested bytrypsinization and replated on laminin-coated plates in neuroepithelialculture medium without the addition of CEE. Omission of CEE was used topromote differentiation. Laminin was used as a substrate instead offibronectin because laminin has, been shown to promote proliferation andneuronal differentiation. J. Drago et al., supra. After 5 days onlaminin-coated plates in NEP medium without CEE, the cells were fixedand processed for determining immunoreactivity to β-III tubulin,neurofilament 160 (NF160), low affinity neurotrophin receptor (p75), andcholine acetyl transferase (ChAT), according to the procedure of Example4. Under these conditions, NEP cells rapidly differentiated, ascharacterized by alterations in morphology and the expression oflineage-specific antigenic markers.

[0059] Small phase bright cells with small processes could be seen asearly as 48 hours after replating onto laminin-coated plates in theabsence of CEE. Cells with this morphology expressed β-III tubulinimmunoreactivity, and a subset of the β-III immunoreactive cells alsoexpressed neurofilament 160 (NF160) immunoreactivity. β-III tubulinimmunoreactive, NF160-negative cells were also observed, and these cellslikely represent immature neurons. S. P. Memberg & A. K. Hall, supra.The number of β-III tubulin immunoreactive cells increased in cultureover a period of 5 days, at which time they represented 20%±4% of thetotal number of cells.

[0060] In addition to the small phase bright, β-III tubulinimmunoreactive cells, cells with a larger cell soma and more elaborateprocesses were also seen. These cells were p75, NF160, and ChATimmunoreactive and were observed both as single cells and as clusters.In the developing neural tube, p75 and ChAT immunoreactivity ischaracteristic of motoneurons. W. Camu & C. E. Henderson, Purificationof Embryonic Rat Motoneurons by Panning on a Monoclona-L Antibody to theLow-affinity NGF Receptor, 44 J. Neurosci. Meth. 59-70 (1992). The p75,ChAT immunoreactive cells (hereinafter, “motoneurons”) represented asmall proportion (4%±2%) of the total number of cells.

EXAMPLE 6

[0061] In this example, NEP cells grown on fibronectin in NEP medium for5 days according to the procedure of Example 1 were harvested bytrypsinization and replated on fibronectin-coated plates in NEP mediumwithout CEE but with the addition of 10% FBS for a period of 5 days.Omission of CEE was used to promote differentiation. The cells were thenfixed and processed for GFAP, p75, nestin, β-III tubulin, and A2B5immunoreactivity, according to the procedure of Example 4. Under theseconditions, NEP cells rapidly differentiated, and the largest proportionof differentiated cells expressed glial fibrillary acid protein (GFAP)immunoreactivity. After 5 days in culture, GFAP immunoreactive cellsconstituted 73%±6% of the total number of cells present. Twocharacteristic morphologies could be identified, a flattened,pancake-shaped cell with small or absent processes, and a smaller, morefibroblastic cell with long, elaborate processes. Neither of these twomorphologically distinct cells expressed A2B5, p75, or β-III tubulinimmunoreactivity, indicating that these cells were most likely type 1astrocytes. No type 2 astrocytes, as defined by co-expression of A2B5and GFAP, M. Raff, Glial Cell Diversification in the Rat Optic Nerve,243 Science 1450-55 (1989); L. E. Lillien & M. C. Raff, Analysis of theCell-cell Interactions that Control Type-2 Astrocyte Development InVitro, 4 Neuron 525-34 (1990), were identified, though such type 2astrocytes have been generated from NEP cells in other cultureconditions (e.g. Examples 8 and 12).

EXAMPLE 7

[0062] In this example, NEP cells grown on fibronectin in NEP medium for5 days according to the procedure of Example 1 were harvested bytrypsinization and replated on laminin-coated plates in neuroepithelialculture (NEP) medium without the addition of CEE for 5-10 days.Differentiating NEP cells were then labeled, according to the procedureof Example 4, with markers previously identified as being expressed onoligodendrocytes and their precursors: A2B5, GalC, O1, and O4. Threedays after replating NEP cells, a subset of the cells began to expressA2B5 immunoreactivity. A2B5 immunoreactive cells initially did notexpress detectable levels of GalC, O4, and O1 immunoreactivity. After anadditional three days in culture, however, GalC immunoreactive cellscould be seen, which cells also expressed A2B5 immunoreactivity. Suchcells appeared flattened and did not have the characteristic morphologyof oligodendrocyte-type 2-astrocyte (O-2A) progenitors or matureoligodendrocytes. Longer periods in culture, however, allowed moremature-looking oligodendrocytes with a small body and extensiveprocesses to develop. These cells expressed O1 and GalCimmunoreactivity, markers characteristic of differentiatedoligodendrocytes. Thus, NEP cells can generate oligodendrocytes thatmature over 10 days in culture. The pattern of antigen expressionfurther suggests the existence of a dividing oligodendrocyte precursorthat subsequently generates oligodendrocytes, as has been described fromspinal cord cultures from older embryos. B. C. Warf et al., supra; R. H.Miller & V. Szigeti, Clonal Analysis of Astrocyte Diversity in NeonatalSpinal Cord Cultures, 115 Development 133-42 (1991).

[0063] Therefore, as shown in Examples 5-7, NEP cells grown in culturegenerate neurons, glia, and oligodendrocytes when replated on laminin inthe absence of CEE. This culture condition, while suboptimal for anyparticular phenotype, is sufficient to generate differentiated progenyand has been used to assess differentiation in subsequent experiments.

EXAMPLE 8

[0064] NEP cells grown in culture could be either a homogeneouspopulation of cells where each cell could differentiate into allphenotypes or a heterogeneous population of cells with a variety ofdifferentiation potentials. To distinguish between these possibilities,cultured NEP cells were grown at clonal density, individual cells werecircled, and their development followed for a period of 15 days. Cloneswere analyzed for differentiation by triple labeling using GFAP, β-IIItubulin, and A2B5 as markers for astrocytes, neurons, andoligodendrocyte precursors.

[0065] For preparation of clonal cultures of neuroepithelial cells, NEPcells prepared according to the procedure of Example 1 were trypsinizedand plated in 35 mm dishes coated with fibronectin at a dilution ofabout 50 cell/dish. In some experiments, however, cells were plated atabout 10 cells/dish. Cells were allowed to settle for a period of 4hours, and then single cells were circled and their development followedin culture. In most experiments, clonal cultures were terminated after12 days. In experiments to demonstrate oligodendrocyte development,clones were observed for 18-21 days.

[0066] For replating individual clones, a glass cloning ring (FisherScientific, Pittsburg, Pa.) was placed around each clone and the cellsisolated by trypsinization for 1-2 minutes with 100 μl of trypsin/EDTAsolution. Cells were resuspended in fresh medium, and an aliquot ofcells (50-100 cells) was replated onto fibronectin-coated culturesdishes. Single cells were identified and circled with a grease penciland their development followed as described above.

[0067] Primary or replated clonal culture plates were usually triplelabeled with the cell surface antigen and the appropriate secondaryantibody being used in live cell culture according to the proceduresdescribed in Example 4. Clones were then fixed in 4% paraformaldehydefor 10 minutes and processed sequentially for the other antigens. Thediaminobenzidine (DAB, Sigma) reaction to horseradish peroxidase labeledsecondary antibodies was always performed after all other staining hadbeen completed because reduced staining with some antigens was observedif the clones were processed for DAB histochemistry first.

[0068] At least some clones were stained by all three markers and thuscontained all three phenotypes of cells. Thus, at least some NEP cellsare capable of generating neurons, astrocytes, and oligodendrocytes. Toconfirm that A2B5 immunoreactive cells represented oligodendrocytes,some clones were restained with O1 or GalC. The results summarized inTable 2 represent 256 colonies from three independent clonal assays.TABLE 2 Antigen Expressed % of Clones A2B5 + β-III tubulin 13 ± 2 A2B5 +GFAP 28 ± 2 A2B5 + β-III tubulin + GFAP 42 ± 3 GFAP + β-III tubulin 17 ±1 GFAP alone None β-III tubulin alone None A2B5 alone None

[0069] All clones analyzed contained more than one phenotype. Neuron andoligodendrocyte clones, as well as neuron and astrocyte clones wereidentified. A significant proportion of NEP cells generated coloniescontaining all three phenotypes of cells. In all cases, when clones werecarefully studied, it was possible to identify cells that did notexpress any of the markers tested, suggesting that precursor cells werestill present. Further, no clones that contained only one cell typecould be identified, suggesting that at this stage no committedprecursors were present in culture.

EXAMPLE 9

[0070] To determine if multipotent stem cells underwent self renewal,NEP cells prepared according to the procedure of Example 1 were platedat low density and single cells were observed for 10 days according tothe procedure of Example 8. Clones at this stage varied in size fromabout 100 to several thousand cells. The largest clones were identified,harvested by trypsinization, and a subset of cells was replated onfibronectin-coated plates in NEP medium. Individual cells from eachparent clone were circled and observed in culture. Fifteen days afterreplating, clones were triple labeled for O1, β-III tubulin, and GFAPexpression. The number of daughter clones that expressed all threemarkers is shown in Table 3, which contains the pooled results fromthree independent NEP cell preparations. TABLE 3 No. of Cells No. ofMultipotent Clone No. Observed Daughter Clones 1 40 15 2 34 3 3 38 12 436 8 5 42 9 6 22 2 7 21 3 8 13 1 9 17 8 10 21 7 11 19 3 12 23 4 13 41 1314 16 7 15 37 9

[0071] Of the 15 clones that were followed, each contained 1-15 daughterclones (3-50% of replated cells) that had differentiated into neurons,astrocytes, and oligodendrocytes. Thus, all of the clones that wereobserved generated multipotent daughter cells. Therefore, individual NEPcells are capable of self renewal.

EXAMPLE 10

[0072] Stem cells that undergo self renewal and retain their ability todifferentiate into multiple phenotypes have been previously described.B. A. Reynolds et al., A Multipotent EGF-responsive Striatal EmbryonicProgenitor Cell Produces Neurons and Astrocytes, 12 J. Neurosci.4565-4574 (1992); B. A. Reynolds et al., Clonal and Population AnalysisDemonstrate that an EGF-responsive Mammalian Embryonic CNS Precursor isa Stem Cell, 175 Develop. Biol. 1-13 (1996); A. L. Vescovi et al., bGFGRegulates the Proliferative Fate of Unipotent (Neuronal) and Bipotent(Neuron/Astroglial) EGF-generated CNS Progenitor Cells, 11 Neuron 951-66(1993); T. J. Kilpatrick & B. F. Bartlett, supra; T. J. Kilpatrick & B.F. Bartlett, Cloned Multipotential Precursors from the Mouse CerebrumRequire FGF-2 whereas Glial Restricted Precursors are Stimulated byeither FGF-2 or EGF, 15 J. Neurosci. 3653-61 (1995); A. A. Davis & S.Temple, A Self Renewing Multipotential Stem Cell in Embryonic RatCerebral Cortex, 372 Nature 263-66 (1994); S. Temple & A. Davis,Isolated Rat Cortical Progenitor Cells are Maintained in Division InVitro by Membrane Associated Factors, 120 Development 999-1008 (1994).One such stem cell is the neurosphere isolated from cortical ventricularzone, which can be maintained in an undifferentiated state over multiplepassages in defined medium in the presence of EGF. B. A. Reynolds et al.(1992), supra; B. A. Reynolds et al. (1996), supra; A. L. Vescovi etal., supra. To determine if NEP cells could be grown as neurospheres,cells grown in adherent cultures according to the procedure of Example 1were trypsinized, pelleted, and grown in bacterial plates as suspensioncultures at a density of 100-300 cells, i.e. in non-adherent culture atclonal density. The medium used was NEP medium. Most cells did notsurvive replating, but, on average, 2.5±1.0 cells (1.2%) formedneurospheres. No neurospheres were obtained when cells were grown in NEPmedium in which EGF (50 ng/ml) was substituted for FGF (20 ng/ml).

[0073] Neurospheres generated in FGF-containing medium were replatedonto either fibronectin-coated dishes in non-differentiating medium oronto laminin-coated plates in differentiating medium (NEP medium minusCEE). Spheres grown on fibronectin were labeled with BrdU and nestin,showing that the majority of cells consisted of undifferentiatednestin-immunoreactive, dividing cells. Such undifferentiated cellsappeared morphologically similar to NEP cells that were generated fromneural tube dissociation, and could be passaged and used to generateadditional neurospheres. Spheres grown on laminin were triple labeledfor O1, β-III tubulin, and GFAP expression, showing that neurospherescan differentiate into neurons, astrocytes, and oligodendrocytes. Thus,NEP cells and FGF-dependent neurospheres represent identical cells grownunder adherent or non-adherent culture conditions, respectively, but aredistinct from the EGF-dependent neurospheres generated from olderembryos.

EXAMPLE 11

[0074] Motoneurons are the earliest cell type to differentiate fromcaudal neuroepithelium. E.g. Hamburger, supra. N-CAM (neural celladhesion molecule) and p75 immunoreactive neurons are seen in vivo andin vitro within 12 hours of the time that neural tubes are isolated andNEP cells placed in culture. E. W. Chen & A. Y. Chiu, supra; W. Camu &C. E. Henderson, supra. It is therefore possible that a committedmotoneuron precursor was already present at the time NEP cells wereplaced in culture. To determine if such a precursor existed, NEP clonalcultures were analyzed with motoneuron and other lineage-specificmarkers. E10.5 NEP cells were isolated and cultured onfibronectin-coated dishes for 5 days, harvested by trypsinization, andreplated onto fibronectin-coated 35 mm dishes at clonal density in NEPmedium with CEE, according to the procedure of Example 8. Singleisolated cells were circled and observed for a period of 10-21 days.Clones were then either (a) double-labeled for ChAT and either β-IIItubulin, GFAP, or A2B5, or (b) triple-labeled for ChAT, β-III tubulin,and A2B5 expression, according to the procedure of Example 4. Cloneswere then scored for the markers they expressed. These results aresummarized in Table 4. TABLE 4 Antigen Expressed Proportion of Clones(%) ChAT + β-III tubulin 26/28 (93%) ChAT + GFAP 30/32 (94%) ChAT + A2B524/27 (89%)

[0075] Table 4 represents the data from 87 clones and shows the numberof clones expressing both markers when double-labeled. No clones wereobserved that contained only ChAT immunoreactive cells, thus no clonecontaining motoneurons alone was observed. Motoneuron-containing clonesalso contained astrocytes, other neurons, and/or oligodendrocytes. Theseresults are evidence, therefore, that there is a common progenitor thatcan generate motoneurons and other spinal cord cells.

Glial Restricted Precursors Derived from NEP Stem Cells

[0076] Multipotent NEP stem cells can be induced to generateself-renewing precursor cells restricted to subsequent glialdifferentiation. The self-renewing precursor population can be isolatedby immunopanning using the monoclonal antibody A2B5 and can bemaintained in an undifferentiated state over multiple divisions whengrown in platelet derived growth factor (PDGF) and bFGF. A2B5⁺ cellsdiffer from parental NEP cells in antigenic phenotype anddifferentiation potential. A2B5⁺ cells lack the ability to differentiateinto neurons under conditions that promote neuronal differentiation inNEP cells. A2B5⁺ cells retain, however, the ability to differentiateinto oligodendrocytes and astrocytes and are thus identified asmultipotential glial-restricted precursors.

[0077]FIG. 2 shows a model of NEP cell differentiation, whereinmultipotent NEP cells 50 have the capability to differentiate into anoligodendrocyte-astrocyte (O-A) progenitor 54 that is capable ofself-renewal and also retains the capability to further differentiateinto oligodendrocytes 58, type 1 astrocytes 62, and type 2 astrocytes66. FIG. 2 also illustrates that NEP cells are capable ofdifferentiating into neurons 70, whereas O-A progenitor cells are not.

[0078] Several lines of evidence show that A2B5 immunoreactiveglial-restricted precursors arise from multipotent NEP cells. First, NEPcells are a homogeneous nestin-positive, A2B5-negative population ofcells (Example 4). Second, clonal analysis of NEP cell cultures revealsno clones that give rise to only glial cells (Example 8). Third, A2B5⁺cells always arise in clones that contain A2B5⁻ neurons and astrocytes(Example 8). Thus, there is no evidence that NEP cells contain acommitted A2B5⁻, O-2A progenitor. Rather, a process of differentiationoccurs where an NEP cell transits to a more restricted cell type.

[0079] The A2B5⁺ population derived from NEP cells appears homogeneousand uniformly lacks the ability to generate neurons. These A2B5⁺ cellsshare some similarities with, but are also different from, other glialrestricted precursors identified in the CNS. F. Aloisi et al.,Developmental Appearance, Antigenic Profile, and Proliferation of GlialCells of the Human Embryonic Spinal cord: An Immunocytochemical StudyUsing Dissociated Cultured Cells, 5 Glia 181-81 (1992); H. M. Blau & S.M. Hughes, Cell Lineage in Vertebrate Development, 2 Curr. Biol. 981-85(1990); R. S. Cameron & P. Rakic, Glial Cell Lineage in Cerebral Cortex:A Review and Synthesis, 4 Glia 124-37 (1991); C. L. Chan et al.,Oligodendrocyte-type 2 Astrocyte (O-2A) Progenitor Cells from Neonataland Adult Rat Optic Nerve Differ in Their Responsiveness toPlatelet-Derived Growth Factor, 55 Brain Res. Dev. Brain Res. 275-82(1990); P. Cochard & M C. Giess, [Oligodendrocyte Lineage], 189 C RSeances Soc. Biol. Fil. 263-69 (1995); A. A. Davis & S. Temple, supra;G. A. Elder et al., Characterization of Glial Subpopulations in Culturesof the Ovine Central Nervous System, 1 Glia 317-27 (1988); J. Fok-Seang& R. H. Miller, Distribution and Differentiation of A2B5⁺ GlialPrecursors in the Developing Rat Spinal Cord, 37 J. Neurosci. Res.219-35 (1994); B. P. Fulton et al., Visualization of O-2A ProgenitorCells in Developing and Adult Rat Optic Nerve by Quisqualate-StimulatedCobalt Uptake, 12 J. Neurosci. 4816-33 (1992); D. S. Galileo et al.,Neurons and Glia Arise from a Common Progenitor in Chicken Optic Tectum:Demonstration with Two Retroviruses and Cell Type-Specific Antibodies,87 Proc. Nat'l Acad. Sci. USA 458-62 (1990); A. L. Gard et al.,Oligodendroblasts Distinguished from O-2A Glial Progenitors by SurfacePhenotype (O4+GalC−) and Response to Cytokines Using Signal TransducerLIFR Beta, 167 Dev. Biol. 596-608 (1995); R. Hardy & R. Reynolds,Proliferation and Differentiation Potential of Rat ForebrainOligodendroglial Progenitors Both In Vitro and In Vivo, 111 Development1061-80 (1991); R. J. Hardy & V. L. Friedrich, Jr., OligodendrocyteProgenitors Are Generated Throughout the Embryonic Mouse Brain, ButDifferentiate in Restricted Foci, 122 Development 2059-69 (1996); P. E.Knapp, Studies of Glial Lineages and Proliferation In Vitro Using anEarly Marker for Committed Oligodendrocytes, 30 J. Neurosci. Res. 336-45(1991); M. B. Luskin et al., Neurons, Astrocytes, and Oligodendrcocytesof the Rat Cerebral Cortex Originate from Separate Progenitor Cells: AnUltrastructural Analysis of Clonally Related Cells, 13 J. Neurosci.1730-50 (1993); R. H. Miller, Oligodendrocyte Origins, 19 TINS 92-96(1996); K. Ono et al., Early Development and Dispersal ofOligodendrocyte Precursors in the Embryonic Chick Spinal Cord, 121Development 1743-54 (1995); M. C. Raff et al., A Glial Progenitor CellThat Develops In Vitro into an Oligodendrocyte Depending on CultureMedium, 303 Nature 390-96 (1983); M. J. Rivkin et al., OligodendroglialDevelopment in Human Fetal Cerebrum, 38 Ann. Neurol. 92-101 (1995); P.M. Wood & A. K. Williams, The Generation of Neurons and Oligodendrocytesfrom a Common Precursor Cell, 7 Neuron 685-93 (1984). NEP-derivedA2B5⁺cells share several characteristics with optic-nerve-derived O-2Aprogenitor cells, including morphology, migratory nature, responsivenessto PDGF and bFGF, and the ability to generate oligodendrocytes andtype-2 astrocytes. In contrast to postnatal O-2A progenitor cells,however, NEP-derived A2B5⁺ cells can also give rise to type 1astrocytes. It thus appears that A2B5⁺ cells purified from NEP cellsrepresent an earlier stage of glial precursor cell development than theA2B5⁺ O-2A progenitor cells that have been studied so extensively.

[0080] Therefore, the presently described invention provides directevidence for a lineage relationship between multipotent andlineage-restricted precursor cell populations and identifiesmorphological, antigenic, and cytokine dependence data to distinguishbetween the two populations. Moreover, there is established anaccessible culture system to follow the development of isolatedprecursor cells and to study the cellular and molecular events thatregulate differentiation processes.

EXAMPLE 12

[0081] Neurons, oligodendrocytes, and astrocytes can be identified usinga variety of lineage specific markers. G. S. Eisenbarth et al.,Monoclonal Antibody to Plasma Membrane Antigen of Neurons, 76 Proc.Nat'l Acad. Sci. USA 4913-17 (1979); E. E. Geisert & A. Frankfurter, TheNeuronal Response to Injury As Visualized by Immunostaining of Classβ-tubulin in the Rat, 102 Neurosci. Lett. 137-41 (1989); I. Sommer & M.Schachner, Monoclonal Antibody (O1-O4) to Oligodendrocyte Cell Surfaces:An Immunocytological Study in the Central Nervous System, 83 Dev. Biol.311-27 (1981); P. A. Trimmer et al., Combination of In SituHybridization and Immunocytochemistry to Detect Messenger RNAs inIdentified CNS Neurons and Glia in Tissue Culture, 39 J. Histochem.Cytochem. 891-98 (1991). Table 5 shows the lineage specific markers usedin the present example. TABLE 5 Antibodies That Identify Specific CellTypes Antibody Antigen Cell Type A2B5 mouse IgM, 1:2 gangliosides Glialprecursors O4 mouse IgM, 1:2 galactoside Oligodendrocytes/ precursorsα-GalC mouse IgG, galactocerebroside Oligodendrocytes 1:2 α-GFAP rabbitIgG, glial fibrillary Astrocytes 1:500 acid α-β-III tubulin intermediateNeurons mouse IgG, 1:400 filament RT-97 mouse IgG, 1:5 neurofilamentNeurons

[0082] In addition to defining differentiated cells, some precursorcells can also be recognized by specific antibodies. Two such markerswere used herein, nestin and A2B5. Nestin is expressed by a variety ofundifferentiated cells in the CNS. E.g., U. Lendahl et al., supra. TheA2B5 antibody labels O-2A progenitor cells.

[0083] In this example, NEP cells prepared from E10.5 rat neural tubecells according to the procedure of Example 1 were grown in the presenceof CEE and bFGF for 3 days and were then replated at 5000cells/coverslip in NEP medium devoid of CEE for an additional 5 days.Cells were incubated for 24 hours with BrdU and stained with anti-BrdUaccording to the procedure of Example 4. Parallel cultures were doublestained after 7 days with selected combinations of the antibodiesdescribed in Table 5. Seventy percent of NEP cells cultured in theabsence of CEE for three days exhibited A2B5 immunoreactivity. TheseA2B5⁺ cells had a flat morphology and were able to divide in thepresence of bFGF. After 4 days in culture in the absence of CEE, 81%±7%of the A2B5⁺ NEP-derived cells were engaged in cell division asdetermined by anti-BrdU immunoreactivity. Double labeling of theNEP-derived A2B5⁺ cells with the antibodies α-nestin, (α-GalC, α-GFAP,α-β--III tubulin, and α-p75 (an antibody against the low affinity NGFreceptor that recognizes a subset of astrocytes) showed that none of thelineage markers were coexpressed on A2B5⁺ cells. A substantial subset ofthe A2B5⁺ cells, however, expressed α-nestin. This coexpression ofα-nestin and A2B5 has been previously described on O-2A progenitorcells. Thus, NEP-derived A2B5-positive cells are antigenically similarto O-2A progenitor cells.

[0084] After an additional 2 days in culture, A2B5⁺ cells had begun toexpress glial-specific markers. A subpopulation of cells was clearlyGalC⁺ by that time. To confirm that cells were sequentiallydifferentiating into oligodendrocytes, cultures were stained with O4 andα-GalC. As expected, 30% of the O4⁺ cells coexpressed α-GalC, resemblingimmature oligodendrocytes. Double labeling with A2B5 and αGFAP showedthat 10% of the A2B5⁺ cells were also GFAP⁺, resembling the antigeniccharacteristic of type-2 astrocytes. All the markers that werecoexpressed at that later time point on a subset of A2B5⁺ cells arecharacteristic of cells belonging to the O-2A lineage. These resultssuggested that at least a subset of the A2B5⁺ cells represented glialprecursor cells and that A2B5 was a useful marker to define thissubpopulation of cells in more detail.

EXAMPLE 13

[0085] To determine whether A2B5⁺ cells arise from multipotent NEP cellsor whether A2B5⁺ cells arise from an already committed subpopulation ofA2B5⁻ NEP cells, NEP cells were plated at clonal densities according tothe procedure of Example 8, and their development in culture wasfollowed for 10 days. Cells were then double stained with the antibodycombinations A2B5/α-β—III tubulin or A2B5/α-GFAP. The results ofanalysis of 132 clones are summarized in Table 6. TABLE 6 Antigenexpressed % of Clones No. of Clones A2B5⁺/β-III tubulin⁺ 93% 71/76A2B5⁺/GFAP⁺ 91% 51/56 A2B5⁺ alone 0  0/132

[0086] Nearly all of the 132 clones consisted of a mixture of A2B5⁺,GFAP⁺, and β-III tubulin⁺ cells. Ninety-one percent of the clonescontained cells that were either A2B5⁺ or GFAP⁺, while 93% of the cloneswere either A2B5⁺ or β-III tubulin⁺. None of the analyzed clonesconsisted only of cells that were A2B5⁺. It is noteworthy that althoughat this early stage none of the clones contained GalC⁺ cells,oligodendrocytes could be identified in clonal cultures and in massculture at later stages (12-15 days after plating on medium devoid ofCEE). These clonal analyses suggest that the A2B5⁺ population arose froma common multipotential A2B5⁻precursor cell.

EXAMPLE 14

[0087] To determine directly whether NEP-derived A2B5⁺ cells can onlygive rise to glial cells, the A2B5⁺ population was purified by specificantibody capture assay (immunopanning). L. J. Wysocki & V. L. Sato,Panning for Lymphocytes: A Method for Cell Selection, 75 Proc. Nat'lAcad. Sci. 2844-48 (1978); M. Mayer et al., Ciliary Neurotrophic Factorand Leukemia Inhibitory Factor Promote the Generation, Maturation, andSurvival of Oligodendrocytes, 120 Development 142-53 (1994), herebyincorporated by reference. Briefly, cells prepared according to Example1 were trypsinized and the suspension was plated on anA2B5-antibody-coated dish to allow binding of all A2B5⁺ cells to theplate. The supernate was removed, and the plate was washed with DMEMsupplemented with additives (DMEM-BS) described by J. E. Bottenstein &G. H. Sato, Growth of Rat Neuroblastoma Cell Line in Serum-FreeSupplemented Medium, 76 Proc. Nat'l Acad. Sci. USA 514-17 (1979), herebyincorporated by reference. The bound cells were scraped off and platedon fibronectin/laminin coated glass coverslips in 300 μl DMEM-BS ±growthfactors at 5000 cells/well. In the final culture, the contaminatingA2B5⁻ cells represented less than 10% of the total cells. The A2B5antibody for coating the plates was used at a concentration of 5 μg/mlprotein. Cells were allowed to bind to the plate for 20-30 minutes in a37° C. incubator. Growth factors were added every other day at aconcentration of 10 ng/ml. Recombinant human PDGF-AA was obtained fromChiron Corporation. Recombinant rat ciliary neurotrophic factor (CNTF)was obtained from Precision Research Biochemicals. Recombinant bFGF waspurchased from PeroTech Inc., and retinoic acid (RA) was from Sigma.

[0088] After 5 days of culturing NEP cells in the absence of CEE, cellswere immunopurified, plated on fibronectin/laminin coated dishes, andexposed to cytokines previously associated with differentiation ofprecursor into oligodendrocytes, astrocytes, or neurons. The A2B5-pannedpopulation was >98% positive for A2B5⁺ cells when stained one hour afterpanning. Staining 24 hours after plating showed that all cells of thepanned population were A2B5⁺ and did not express any other lineagemarkers tested.

[0089] Panned cultures in the presence of bFGF and no other growthfactors for 5 days consisted of 1% oligodendrocytes, 50% GFAP⁺astrocytes, and 49% A2B5⁺ cells. The proportion of differentiated cellswas significantly shifted when the bFGF-containing medium was replacedafter 3 days with medium supplemented only with PDGF. Under theseconditions, the culture consisted of 30% oligodendrocytes, 50%astrocytes, and 20% A2B5⁺ cells.

[0090] Although growth in the presence of bFGF alone was sufficient toallow differentiation of NEP cells into neurons in the parentpopulation, no neurons were detected in the A2B5⁺ panned populationcultured in the presence of bFGF. To enhance the probability of neuronaldifferentiation, the medium was additionally supplemented with retinoicacid, which significantly increased neuronal differentiation in theparent NEP cell population. Even in this neuron-promoting environment,the immunopurified A2B5⁺ population did not contain β-III tubulin⁺cells. It was unlikely that the neuronal population was lost throughselective cell death, because no significant cell death was observed inthe panned mass cultures at any time, suggesting that neurons did notappear rapidly and die. Moreover, no evidence of β-III tubulin⁺ ghostswas detected.

[0091] These results suggest that the precursor cells that areresponsible for generating neurons were not part of the immunopurifiedA2B5⁺ population. As the A2B5-panned cells gave rise to astrocytes andoligodendrocytes, but not to neurons, it appeared that the A2B5⁺population contained precursor cells that were restricted to gliallineage.

EXAMPLE 15

[0092] Mass culture experiments suggested that the A2B5-pannedpopulation prepared according to Example 14 contained cells with adifferentiation potential restricted to glial lineages. This experiment,however, did not address whether astrocytes and oligodendrocytes aregenerated from committed unipotential cells present in the A2B5⁺population or whether single cells are bipotential and can generate bothastrocytes and oligodendrocytes. To address this question, clonalexperiments were performed, wherein the A2B5-panned population wasstained with A2B5 1 day after panning, and cells were plated at limitingdilution in 96-well plates. Wells were scored with immunofluorescence,and wells with one A2B5⁺ stained cell were recorded and cultured inPDGF/bFGF for 7 days. This procedure allowed the expansion of clones andalso minimized the amount of cell death occurring when single cells weredirectly plated into differentiation conditions. After 7 days, expandedclones contained from 50-200 cells and were uniformly A2B5⁺.

[0093] The majority of the clones (51) were first washed with bFGF-freeDMEM-BS and then switched to PDGF-supplemented medium, an effectiveculture condition to induce oligodendrocyte generation, as shown in massculture experiments. All clones contained oligodendrocytes, GFAP⁺astrocytes, and A2B5⁺ cells, while none of the clones contained β-IIItubulin⁺ cells, suggesting that single A2B5⁺ cells were at leastbipotential and also were restricted to glial cell lineages (Table 7).TABLE 7 Growth Condition Marker expressed PDGF FGF/CNTF A2B5⁺/CFAP⁺ 0 6A2B5⁺ 51 6 GFAP⁺ 51 4 GalC⁺ 51 1 β-III tubulin⁺ 0 0 Total No. of Clones51 6

[0094] The differentiation potential of A2B5⁺ cells in a culture mediumsupplemented with bFGF and CNTF was also tested. From the panned massculture experiments it seemed clear that bFGF alone leads to an increasein the number of GFAP⁺ astrocytes and a decrease in the number ofoligodendrocytes. Depending on culture conditions, CNTF has been shownto promote oligodendrocyte generation, M. Mayer et al., supra, or tolead to the generation of type-2 astrocytes, which are GFAP⁺ andtransiently express A2B5. L. E. Lillien & M. C. Raff, DifferentiationSignals in the CNS: Type-2 Astrocyte Development In Vitro as a ModelSystem, 5 Neuron 5896-6273 (1990). Six clones were analyzed that wereexpanded in PDGF/bFGF and then switched to bFGF/CNTF. Surprisingly, allsix clones contained cells that were A2B5⁺/GFAP⁺, resembling the type-2astrocyte phenotype. Only 1 clone contained GalC⁺ oligodendrocytes, andno clone contained β-III tubulin⁺ cells. This result suggested that inthe presence of CNTF and bFGF, A2B5⁺ cells predominantly differentiateinto cells with a type-2 astrocyte phenotype.

[0095] Five A2B5⁺ clone were analyzed in different neuron-promotingconditions and, as before, were unable to generate neurons. FivePDGF/bFGF expanded clones were trypsinized, divided into two portionsand replated into either bFGF alone or bFGF supplemented with retinoicacid. Clones were stained with the antibodies A2B5, α-GFAP, α-GalC, andα-β-III tubulin (Table 8). TABLE 8 Growth Condition Marker expressed FGFFGF/RA A2B5⁺/GFAP⁺ 0 0 A2B5⁺ 5 5 GFAP⁺ 5 5 GalC⁺ 1 0 β-III tubulin⁺ 0 0Total No. of Clones 5 5

[0096] None of the clones, regardless of whether cells were grown inbFGF alone or bFGF/RA, contained β-III tubulin immunoreactivity. Incontrast, all five clones consisted of a mixture of cells that wereeither A2B5⁺ or GFAP⁺, but not both. Only one clone grown in bFGF alonecontained GalC immunoreactive oligodendrocytes, whereas in bFGF/RA, noGalC⁺ oligodendrocytes were found. These data support the initialobservation, that A2B5⁺ cells isolated from induced NEP cell cultureswere multipotential and restricted in their differentiation potential tocells of the glial lineages.

EXAMPLE 16

[0097] To fulfill the criteria of a true intermediate precursor, cellsneed to have an extended self-renewal capacity without losing theability to differentiate into more than one specific cell type. To testthe self-renewal capacity of individual A2B5⁺ cells, two clones expandedin PDGF/bFGF for 7 days were selected for long-term culture andpassaging. The two clones were refed every other day with PDGF/bFGF andmaintained for a total of 3 months with 4 serial passages. Clones weregrown in PDGF/bFGF, as this combination of cytokines apparentlyinhibited differentiation and promoted division. Cells were stainedbefore and after each passage and were negative for all differentiationmarkers tested, except for A2B5⁺, at all time points.

[0098] To determine the differentiation potential of long term clones,during each passage single cells were replated, re-expanded to 50-200cells, and switched to PDGF alone to promote differentiation. In thesesecondary cultures, oligodendrocytes and astrocytes appearedconsistently after 8-10 days. The ability to differentiate intooligodendrocytes and astrocytes was not altered significantly withincreased passages, suggesting that these long-term propagated cellswere still multipotential.

[0099] These results show that A2B5⁺ cells that differentiate frommultipotent NEP cells can be expanded and propagated as precursor cells.Passaged individual A2B5⁺ cells self renew and are able to generateoligodendrocytes, A2B5⁺ and A2B5⁻ astrocytes, but not neurons.NEP-derived A2B5⁺ cells thus represent multipotential intermediateprecursor cells restricted to glial lineages.

[0100] All of the references cited herein and not previously expresslyincorporated by reference are hereby incorporated by reference.

What is claimed is:
 1. An isolated, pure population of mammalian CNSneuroepithelial stem cells wherein said cells are capable ofself-renewal in adherent feeder-cell-independent culture medium and ofdifferentiation to CNS neuronal or glial cells.
 2. The population ofclaim 1 wherein said neuroepithelial stem cells express nestin, but donot express polysialated neural cell adhesion molecule, glial fibrillaryacidic protein, sulfatide, neurofilament, choline acetyl transferase,intermediate filament, ganglioside, or galactocerebroside.
 3. Thepopulation of claim 1 wherein said CNS neuronal cells expressintermediate filament and neurofilament
 68. 4. The population of claim 3wherein said CNS neuronal cells express choline acetyl transferase. 5.The population of claim 1 wherein said CNS glial cells express glialfibrillary acidic protein.
 6. The population of claim 5 wherein said CNSglial cells express ganglioside.
 7. The population of claim 1 whereinsaid CNS glial cells express ganglioside.
 8. The population of claim 7wherein said CNS glial cells express sulfatide.
 9. The population ofclaim 7 wherein said CNS glial cells express galactocerebroside.
 10. Thepopulation of claim 1 wherein said neuroepithelial stem cells arefurther capable of differentiation to glial-restricted precursor cells.11. The population of claim 10 wherein said glial-restricted precursorcells are capable of self-renewal in adherent feeder-cell-independentculture medium and capable of differentiation to CNS glial cells but notto CNS neuronal cells.
 12. The population of claim 11 wherein saidglial-restricted precursor cells express nestin and ganglioside, but donot express glial fibrillary acidic protein, sulfatide, orgalactocerebroside.
 13. The population of claim 11 wherein said CNSglial cells express ganglioside and glial fibrillary acidic protein. 14.The population of claim 11 wherein said CNS glial cells express glialfibrillary acidic protein but do not express ganglioside.
 15. Thepopulation of claim 11 wherein said CNS glial cells expressgalactocerebroside but do not express ganglioside.
 16. An isolated, purepopulation of mammalian CNS glial-restricted precursor cells, whereinsaid glial-restricted precursor cells are capable of self-renewal inadherent feeder-cell-independent culture medium and capable ofdifferentiation to CNS glial cells but not to CNS neuronal cells. 17.The population of claim 16 wherein said glial-restricted precursor cellsexpress nestin and ganglioside, but do not express glial fibrillaryacidic protein, sulfatide, or galactocerebroside.
 18. The population ofclaim 16 wherein said CNS glial cells express ganglioside and glialfibrillary acidic protein.
 19. The population of claim 16 wherein saidCNS glial cells express glial fibrillary acidic protein but do notexpress ganglioside.
 20. The population of claim 16 wherein said CNSglial cells express galactocerebroside but do not express ganglioside.21. A method of isolating a pure population of mammalian CNSneuroepithelial stem cells wherein said cells are capable ofself-renewal in feeder-cell-independent adherent culture medium and ofdifferentiation to CNS neuronal or glial cells, comprising the steps of:(a) removing a neural tube from a mammalian embryo at a stage ofembryonic development after closure of the neural tube but prior todifferentiation of cells in the neural tube; (b) dissociating cellscomprising the neural tube removed from the mammalian embryo; (c)plating the dissociated cells in feeder-cell-independent culture on asubstratum and in a medium configured for supporting adherent growth ofthe neuroepithelial stem cells comprising effective amounts offibroblast growth factor and chick embryo extract; and (d) incubatingthe plated cells at a temperature and in an atmosphere conducive togrowth of the neuroepithelial stem cells.
 22. The method of claim 21wherein said mammalian embryo is selected from the group consisting ofprimates, equines, canines, felines, bovines, porcines, ovines, andlagomorphs.
 23. The method of claim 21 wherein said substratum comprisesfibronectin.
 24. The method of claim 21 wherein temperature is about 37°C. and said atmosphere comprises about 5% CO₂ and about 95% air.
 25. Themethod of claim 21 wherein said medium comprises NEP medium.
 26. Amethod of isolating a pure population of mammalian CNS glial-restrictedprecursor cells wherein said cells are capable of self-renewal inadherent feeder-cell-independent culture medium and of differentiationto CNS glial cells but not CNS neuronal cells, comprising the steps of:(a) isolating a population of mammalian CNS neuroepithelial stems cells;(b) incubating the neuroepithelial stem cells in a medium lacking aneffective amount of chick embryo extract for a period of time sufficientfor the cells to begin differentiating; (c) subjecting the incubatedcells to specific antibody capture using an antibody characteristic ofglial-restricted precursor cells to result in a captured subpopulationof cells; and (d) incubating the captured subpopulation of cells in amedium configured for supporting adherent growth thereof comprisingeffective amounts of fibroblast growth factor and platelet derivedgrowth factor.
 27. The method of claim 26 wherein said isolating apopulation of CNS neuroepithelial stem cells comprises: (1) removing aneural tube from a mammalian embryo at a stage of embryonic developmentafter closure of the neural tube but prior to differentiation of cellsin the neural tube; (2) dissociating cells comprising the neural tuberemoved from the mammalian embryo; (3) plating the dissociated cells infeeder-cell-independent culture on a substratum and in a mediumconfigured for supporting adherent growth of the neuroepithelial stemcells comprising effective amounts of fibroblast growth factor and chickembryo extract; and (4) incubating the plated cells at a temperature andin an atmosphere conducive to growth of the neuroepithelial stem cells.28. The method of claim 27 wherein said mammalian embryo is selectedfrom the group consisting of primates, equines, canines, felines,bovines, porcines, ovines, and lagomorphs.
 29. The method of claim 27wherein said substratum comprises fibronectin.
 30. The method of claim27 wherein temperature is about 37° C. and said atmosphere comprisesabout 5% CO₂ and about 95% air.
 31. A method of generating a populationof mammalian motoneurons comprising the steps of: (a) isolating apopulation of mammalian CNS neuroepithelial stems cells; and (b)incubating the neuroepithelial stem cells in a medium that promotes cellproliferation and neuronal differentiation for a period of timesufficient for the cells to begin differentiating.
 32. The method ofclaim 31 wherein the medium comprises laminin-coated plates and NEPmedium lacking an effective amount of chick embryo extract.